Multifunctional enzymes

Wakil, S., 1989. Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry 28 4523-4530. 

In prokaryotes, each reaction of Figure 34-2 is catalyzed by a different polypeptide. By contrast, in eukaryotes, the enzymes are polypeptides with multiple catalytic activities whose adjacent catalytic sites facilitate channeling of intermediates between sites. Three distinct multifunctional enzymes catalyze reactions 3, 4, and 6, reactions 7 and 8, and reactions 10 and 11 of Figure 34-2. 

Auldridge, M. E., A. Block et al. (2006). Characterization of three members of the arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family. Plant J. 45(6) 982-993. 

By 1960 it was clear that acetyl CoA provided its two carbon atoms to the to and co—1 positions of palmitate. All the other carbon atoms entered via malonyl CoA (Wakil and Ganguly, 1959 Brady et al. 1960). It was also known that 3H-NADPH donated tritium to palmitate. It had been shown too that fatty acid synthesis was very susceptible to inhibition by p-hydroxy mercuribenzoate, TV-ethyl maleimide, and other thiol reagents. If the system was pre-incubated with acetyl CoA, considerable protection was afforded against the mercuribenzoate. In 1961 Lynen and Tada suggested tightly bound acyl-S-enzyme complexes were intermediates in fatty acid synthesis in the yeast system. The malonyl-S-enzyme complex condensed with acyl CoA and the B-keto-product reduced by NADPH, dehydrated, and reduced again to yield the (acyl+2C)-S-enzyme complex. Lynen and Tada thought the reactions were catalyzed by a multifunctional enzyme system. 

The first step is carboxylation of acetyl CoA to malonyl CoA. This reaction is catalyzed by acetyl-CoA carboxylase [5], which is the key enzyme in fatty acid biosynthesis. Synthesis into fatty acids is carried out by fatty acid synthase [6]. This multifunctional enzyme (see p. 168) starts with one molecule of ace-tyl-CoA and elongates it by adding malonyl groups in seven reaction cycles until palmi-tate is reached. One CO2 molecule is released in each reaction cycle. The fatty acid therefore grows by two carbon units each time. NADPH+H is used as the reducing agent and is derived either from the pentose phosphate pathway (see p. 152) or from isocitrate dehydrogenase and malic enzyme reactions. 

In the vertebrates, biosynthesis of fatty acids is catalyzed by fatty add synthase, a multifunctional enzyme. Located in the cytoplasm, the enzyme requires acetyl CoA as a starter molecule. In a cyclic reaction, the acetyl residue is elongated by one C2 unit at a time for seven cycles. NADPH+H is used as a reducing agent in the process. The end product of the reaction is the saturated Cie acid, palmitic acid. 

Fatty acid synthase in vertebrates consists of two identical peptide chains—i. e., it is a homodimer. Each of the two peptide chains, which are shown here as hemispheres, catalyzes all seven of the partial reactions required to synthesize palmitate. The spatial compression of several successive reactions into a single multifunctional enzyme has advantages in comparison with separate enzymes. Competing reactions are prevented, the individual reactions proceed in a coordinated way as if on a production line, and due to low diffusion losses they are particularly ef dent. 

The biosynthetic pathway described above indicates the possible involvement of multifunctional enzyme con5>lexes in the biosynthesis of the peptide part of bleomycin. The peptide peurt of bleomycin has no cytotoxicity. Probably, in the final step of the biosynthesis, the disaccharide peirt is transferred to the B-hydroxyl group of the B-l droxylhistidyl moiety and the bleomycin thus synthesized is rapidly released extracellularly. 

Compounds like lev ptln acid, which are the precursors of secondary metabolites, should not have cytotoxicity, and inmany cases, the enzyme involved in the last step of the biosynthesis may be inhibited by the last product as in the case of leupeptin acid reductase. It is also possible that multifunctional enzyme conplexes may be involved in the synthesis of many secondcury metabolites. 

Additionally, a vast array of natural lipopeptides with remarkable structural diversity is produced by microorganisms living in different habitats, from aquatic to terrestrial environments. These products are not gene encoded, but are synthesized nonribosomally by large multifunctional enzymes, the peptide synthetases. 6"8 ... 

The biosynthesis of many hydroxylated natural products proceeds through regio- and enantioselective modification of polyketides, which are assembled through chain elongation via acetate or propionate units [2]. The enzymes responsible for the chain elongation and subsequent reduction, elimination, aromatiza-tion, and further modifications are classified as polyketide synthases [3]. These multifunctional enzymes have been used for whole-cell biotransformation toward unnatural metabolites that are within the scope of combinatorial biosynthesis... 

Like all other retroviruses, human immunodeficiency virus type 1 (HIV-1) contains the multifunctional enzyme reverse transcriptase (RT). Retroviral RTs have a DNA polymerase activity that can use either an RNA or a DNA template and an RNase H activity. HIV-1 RT is essential for the conversion of single-stranded viral RNA into a linear double-stranded DNA that is subsequently integrated into the host cell chromosomes [1-4]. In this conversion process HIV-1 RT catalyzes the incorporation of approximately... 

Chen, M.C. Walker, J. Prusoff, W.H. Kinetic studies of herpes simplex virus type 1-encoded thymidine and thymidylate kinase, a multifunctional enzyme. J. Biol. Chem., 254, 10747-10753 (1979)... 

Perham, R.N. (2000) Swinging arms and swinging domains in multifunctional enzymes catalytic machines for multistep reactions. Annu. Rev. Biochem. 69, 961-1004. 

C. Fatty acid synthase a multifunctional enzyme in eukaryotes... 

A closely related E. coli protein is a 79-kDa multifunctional enzyme that catalyzes four different reactions of fatty acid oxidation (Chapter 17). The amino-terminal region contains the enoyl hydratase activity.32 A quite different enzyme catalyzes dehydration of thioesters of (3-hydroxyacids such as 3-hydroxydecanoyl-acyl carrier protein (see Eq. 21-2) to both form and isomerize enoyl-ACP derivatives during synthesis of unsaturated fatty acids by E. coli. Again, a glutamate side chain is the catalytic base but an imidazole group of histidine has also been implicated.33 This enzyme is inhibited irreversibly by the N-acetylcysteamine thioester of 3-decynoic acids (Eq. 13-8). This was one of the first enzyme-activated inhibitors to be studied.34... 

The previous enzymes catalyze sequential steps in biosynthetic pathways. There are other multifunctional enzymes that have multiple activities for other... 

As discussed briefly in Section I,A, glucose-6-phosphatase is now known to be a multifunctional enzyme capable of catalyzing potent phosphotransferase as well as phosphohydrolase reactions [see Eqs. (1)—(4) ]. Compounds demonstrated to function as effective phosphoryl donors include fructose-6-P (30), mannose-6-P (40), PPi (35-38), a variety of nucleosidetriphosphates and nucleosidediphosphates—most effectively CTP, CDP, deoxy-CTP, ATP, ADP, GTP, GDP, and ITP (41, 45)— carbamyl-P (43), phosphoramidate (44), phosphopyruvate (42, 43) and glucose-6-P itself (30, 31). The various phosphoryl donors are also hydrolyzed by action of the enzyme (see preceding references). Eqqa-tions (1)—(4), which describe these various activities, are given in Section I,A. 

Evidence for the involvement of a single, multifunctional enzyme— microsomal glucose-6-phosphatase—in the above variety of hydrolytic and synthetic reactions has been summarized recently by the author (10) as follows ... 

The metabolic roles and regulation of glucose-6-P phosphohydrolase activity have been considered in detail in reviews by Cahill et al. (6) and Ashmore and Weber (7). More recently, possible metabolically important roles for phosphotransferase activities of this enzyme in liver, kidney, and intestine have been described in reviews by the present author (9, 10)—who also considered a variety of regulatory features based on interaction of substrates, inhibitors, and activators with the multifunctional enzyme—and by Cohn et al. (11). 

Wakil, S. J., Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry 28 4523, 1989. Reviews the evidence for the current model of the mammalian fatty acid synthase as depicted in figure 18.14. 

Ros Barcelo A, Pomar F, Lopez-Serrano M, Pedreno MA. 2003. Peroxidase a multifunctional enzyme in grapevines. Func Plant Biol 30 577-591. 

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